Intact T cell function is critical for effective defense against cryptococcosis. However, the antigen(s) on Cryptococcus neoformans that stimulate a protective cell-mediated (CMI) response against cryptococcosis are poorly characterized. The central hypothesis of this proposal is that there are immunodominant antigens on C. neoformans that stimulate a protective CMI response and that identification of these antigens will allow development of a vaccine to protect individuals at risk from getting cryptococcosis. A novel approach to identifying cryptococcal protein antigens is proposed: murine T cell hybridomas reactive with cryptococcal proteins will be identified and the hybridomas used as tools to purify the antigens. Recombinant proteins will be generated and tested as vaccine candidates in murine models of cryptococcosis. There are three interrelated specific aims: Aim 1. To generate murine T-cell hybridomas reactive with C. neoformans antigens. Following immunization of mice with C. neoformans, splenic T cells will be isolated and fused with thymoma cells. The resulting hyrbidomas will be screened for reactivity with cryptococcal proteins and cloned. Aim 2. To characterize the cryptococcal antigens responsible for stimulating the hybridomas. Each hybridoma clone generated in specific aim 1 will have a unique T-cell receptor reactive with a cryptococcal protein. Using techniques in molecular biology and protein chemistry, the proteins that react with the hybridomas will be identified. cDNA encoding for the proteins of interest will be cloned and recombinant protein isolated. Aim 3. To test the recombinant proteins as vaccine candidates. Mice will be immunized with the recombinant proteins generated in specific aim 2 and then challenged with C. neoformans using intratracheal and intravenous models of infection.